HUMAN JC VIRUS NUCLEAR FACTOR-1 BINDING MOTIFS AND LARGE TUMOR-ANTIGEN REGION REQUIRED FOR TRANSACTIVATION OF LATE PROMOTER

The nuclear factor 1 (NF-1) motifs, NF-1 II/III, in the two 98-bp repe ats of the transcription-regulatory region of JC virus (JCV), have a c ritical role in brain-specific transcription from the JCV early promot er-enhancer, In this study, the role of these motifs in transactivatio n of the JCV late promoter-enhancer (JCV(L)) was examined in different iating glial P19 embryonal carcinoma cells, The expression of papovavi ral large tumor antigen (T-Ag) in the glial cells was shown by double immunofluorescence assays. By using site-directed mutagenesis and in v ivo assays, the two wild-type NF-1 II/III sites, but not the third sit e, were found to be essential for the transactivation of JCV(L) by JCV T-Ag. In vitro transcription assays confirmed this specific transacti vation and the transactivation was abolished by T-Ag antibody, In elec trophoretic mobility shift assays, expression of JCV T-Ag increased th e binding of a factor(s) to the 98-bp repeat. T-Ag antibody abolished the increase of binding. Binding assays with oligonucleotides of NF-1 II/III motifs showed that the increased binding specifically required the wildtype NF-1 II/III sequences and confirmed the requirement of T- Ag. To determine the region of T-Ag necessary for transactivation of J CV(L), the coding sequences were mutated. The amino-terminal region of JCV Ag in amino acids 1-437 was essentially required for efficient tr ansactivation. These results indicated that transactivation of JCV(L) and increased binding require a factor(s) found specifically in glial cells, the JCV NF-1 II/III sites, and the T-Ag amino-terminal region.